Endolysins Active Against Staphylococcus Bacteria, Pharmaceutical Compositions, and Methods Relating Thereto

ABSTRACT

The present invention relates to methods of treating or preventing a bacterial disease or infection, antibacterial compositions, and antibacterial surfaces, including isolated endolysin polypeptides from bacteriophage GRCS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/794,186, filed Jul. 8, 2015, which application is based on U.S.Provisional Patent Application Ser. No. 62/023,096, filed Jul. 10, 2014,which applications are incorporated herein by reference in theirentireties and to which priority is claimed.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported by the U.S. Department of Defense as providedfor by the terms of Contract No. W81XWH1120006 (Grant No. DM102823). TheU.S. government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(file name: 2105_0062C_SUB_SeqList_ST25.txt, created Oct. 21, 2015, andhaving a size of 10,715 bytes), which file is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of treating or preventingbacterial infection, antibacterial compositions, and devices includingantibacterial surfaces, incorporating isolated endolysin polypeptidesfrom bacteriophage GRCS.

BACKGROUND OF THE INVENTION

It has been estimated that 70% of the bacteria that causehospital-acquired infections are now resistant to one or moreantibiotics (Taubes G (2008) The bacteria fight back. Science321(5887):356-61). One of the most alarming antibiotic-resistantbacterial species is Staphylococcus aureus. Specifically,methicillin-resistant S. aureus (MRSA) are the group of S. aureusstrains resistant to the entire class of β-lactam antibiotics.Hospital-acquired MRSA (HA-MRSA) often leads to severe andlife-threatening infections, such as those at surgical sites, in thebloodstream, or pneumonia, while community-acquired MRSA (CA-MRSA)typically leads to superficial skin infections that can ultimatelyprogress to induce severe invasive complications, such as necrotizingfasciitis (Lowy F D (1998) Staphylococcus aureus infections. N Engl JMed 339(8):520-32; Tang Y W & Stratton C W (2010) Staphylococcus aureus:An old pathogen with new weapons. Clin Lab Med 30(1):179-208). In somecases, individuals have died within two days of infection due to theineffectiveness of present-day antibiotics (Romero-Vivas et al. (1995)Mortality associated with nosocomial bacteremia due tomethicillin-resistant Staphylococcus aureus. Clin Infect Dis21(6):1417-23).

Approval of new antibiotics, including linezolid (oxazolidinone class)in 2000, daptomycin (cyclic lipopeptide class) in 2003, and tigecycline(glycylcycline class) in 2005, provides alternatives to vancomycin,which was formerly the only antibiotic treatment for MRSA (Micek S T(2007) Alternatives to vancomycin for the treatment ofmethicillin-resistant Staphylococcus aureus infections. Clinicalinfectious diseases: an official publication of the Infectious DiseasesSociety of America 45 Suppl 3:S184-90). These new antibiotics, alongwith increased awareness and adherence to universal decolonizationpractices, have led to a decrease in the incidence of MRSA in intensivecare units (Huang S S et al. (2013) Targeted versus universaldecolonization to prevent ICU infection. N Engl J Med 368(24):2255-65).Nonetheless, a recent report from the Centers for Disease Control andPrevention indicates there are still over 80,000 severe MRSA infectionsper year in the United States, resulting in over 11,000 deaths (e.g.,see CDC (2013) Antibiotic resistance threats in the United States, 2013.Centers for Disease Control and Prevention, Atlanta). The same CDCreport labeled MRSA as a “serious” public health threat, andvancomycin-resistant S. aureus (VRSA) as a “concerning” threat,underscoring the need for development of alternative therapeutics.

To counteract bacterial resistance and ameliorate the problems caused byS. aureus infections, endolysin therapy is one avenue that is beingpursued (Borysowski J et al. (2011) Potential of bacteriophages andtheir lysins in the treatment of MRSA: current status and futureperspectives. BioDrugs 25(6):347-55; Nelson D C et al. (2012) Endolysinsas antimicrobials. Adv Virus Res 83:299-365). Endolysins are enzymesreleased by bacteriophages during the lytic cycle of viral infection. Alytic enzyme is capable of specifically cleaving bonds that are presentin the peptidoglycan of bacterial cells to disrupt the bacterial cellwall. The bacterial cell wall peptidoglycan is highly conserved amongmost bacteria, and cleavage of only a few bonds is believed to disruptthe bacterial cell wall. Once produced within the bacterial cytoplasm byreplicating bacteriophage, endolysins hydrolyze bonds in the bacterialcell wall (i.e. peptidoglycan) until lysis is complete.

The idea of utilizing endolysins therapeutically is based on thephenomenon of “lysis from without”, a phrase used to describe thedestruction of the bacterial envelope without production of phagevirions (Abedon S T (2011) Lysis from without. Bacteriophage1(1):46-49). This phenomenon only occurs in Gram-positive organisms,such as MRSA, because such bacteria lack an outer membrane protectingthe cell wall (Schmelcher et al. (2011) Domain shuffling and moduleengineering of Listeria phage endolysins for enhanced lytic activity andbinding affinity. Microb Biotechnol 4(5):651-62). Rather, the cell wallof such Gram-positive bacteria includes interconnecting layersconsisting primarily of peptidoglycan. Gram-positive bacteria include,inter alia, numerous species within the genera Actinomyces, Bacillus,Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus,Mycobacterium, Corynebacterium, and Clostridium.

The classical structure of endolysins that act on Gram-positive cellwalls employs a modular architecture consisting of an N-terminalcatalytic domain linked to a C-terminal cell wall binding domain (CBD).The catalytic domain is responsible for cleaving specific covalent bondsin the peptidoglycan structure that are essential for maintaining itsintrinsic structural integrity. The CBD confers endolysin specificity byrecognizing and noncovalent binding to species- or strain-specificepitopes associated with the cell envelope. It is the high specificityderived by the combined actions of the catalytic and CBD domains thatcause endolysins to be highly refractory to the resistance commonlyobserved upon treatment with classical antibiotics (Fischetti V A (2005)Bacteriophage lytic enzymes: novel anti-infectives. Trends Microbiol13(10):491-6; Schuch R et al. (2002) A bacteriolytic agent that detectsand kills Bacillus anthraces. Nature 418(6900):884-9). This is due tothe evolution of bacteriophage to target specific, conserved bonds inthe peptidoglycan of a bacteria cell wall, ensuring that the progenyphage will survive (Low L Y et al. (2011) Role of net charge oncatalytic domain and influence of cell wall binding domain onbactericidal activity, specificity, and host range of phage lysins. JBiol Chem 286(39):34391-403). However, if resistance were to develop,endolysins could be engineered through domain shuffling or used incombination with other endolysins or antibiotics to prolong the use ofthese enzymes (Shen Y et al. (2012) Phage-based Enzybiotics. In: AbedonS, Hyman P (eds) Bacteriophages in Health and Disease. CABI Press, pp217-239).

Thus, identified endolysins have been shown to be effective in killingspecific bacterial strains. However, there still exists a need foradditional and/or alternative endolysin-based therapeutics, particularlytherapeutics exhibiting superior activity and/or which target otherbacterial strains as compared to known therapeutics.

SUMMARY OF THE INVENTION

The increasing rate of resistance of pathogenic bacteria, such as S.aureus, to classical antibiotics has driven research towardsidentification of other means to fight infectious diseases. The presentinvention relates to methods of treating such infectious diseases byadministering to a subject a therapeutically effective amount ofparticular bacteriophage-encoded peptidoglycan hydrolase, calledendolysin(s) or enzybiotic(s). The endolysin polypeptides of the presentinvention lyse the bacterial cell wall upon direct contact, are notinhibited by traditional antibiotic resistance mechanisms, and thus aresuitable for numerous applications, e.g., such as in the areas of foodsafety, human health, and veterinary science.

In particular, the present invention is directed to methods,compositions and devices incorporating or utilizing particular endolysinpolypeptide(s) from the bacteriophage GRCS, sometimes referred to hereinas PlyGRCS endolysin or endolysin polypeptide (which have the disclosedamino acid sequences, variants thereof, or active fragments thereof).According to disclosed embodiments, PlyGRCS endolysin(s) are utilizedfor treating infectious disease associated with Gram-positive bacteria,in particular Staphylococcus bacteria (e.g., S. aureus and S.epidermidis) including methicillin- and vancomycin-resistant strains(e.g., methicillin-resistant S. aureus (MRSA),vancomycin-intermediate-resistant S. aureus (VISA), andmethicillin-resistant S. epidermidis).

Thus, one aspect of the present invention provides for endolysinpolypeptides having killing activity against gram-positive bacteria,particularly Staphylococcus bacteria. In accordance with disclosedembodiments, a method of killing gram-positive bacteria is provided bycontacting bacteria with a composition comprising an amount of isolatedendolysin polypeptide effective to kill such bacteria, the isolatedendolysin polypeptide comprising an amino acid sequence of SEQ ID NOs:5, 6 and/or 7 or variants thereof.

Another aspect of the present invention relates to methods of treatingbacterial infection (e.g., an infection or disease caused by aStaphylococcus species such as S. aureus) in a subject (e.g., a humanpatient) comprising administering to the patient a therapeuticallyeffective amount of an isolated endolysin polypeptide comprising theamino acid sequence of SEQ ID NOs: 5, 6 and/or 7, or variants thereofhaving at least 80% identity thereto. In some embodiments, the methodincludes the further step of administering to the subject a secondarytherapeutic agent (e.g., one or more antibiotic) after or concurrentwith the administration of the isolated polypeptide.

The present invention also relates to pharmaceutical compositions forkilling Gram-positive bacteria comprising an isolated polypeptidecomprising the amino acid sequence of SEQ ID NOs: 5, 6 and/or 7, orvariants thereof having at least 80% identity thereto, and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition further comprises one or more antibiotic,such as for example a penicillin, a cephalosporin, a polymyxin, anansamycin, a quinolone, a sulfonamide, a lipopeptide, a glycycline,and/or an oxazolidinone. In some implementations, the antibiotic isselected from the group consisting of linezolid, daptomycin, andtigecycline, vancomycin, fidaxomicin, and metronidazole.

The present invention is also directed to a substrate (e.g., such as adevice or apparatus, such as a medical instrument or device or animplantable medical device) including a surface comprising anantibacterial coating or material coupled thereto, wherein the coatingor material comprises an isolated polypeptide comprising the amino acidsequence of SEQ ID NOs: 5, 6 and/or 7, or variants thereof having atleast 80% identity thereto. In some implementations, the coating ormaterial comprises one or more secondary therapeutic agent(s), such asfor example an antimicrobial or an antibiotic (e.g., a penicillin, acephalosporin, a polymyxin, an ansamycin, a quinolone, a sulfonamide, alipopeptide, a glycycline, and an oxazolidinone).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates biochemical characterization of optimal conditionsfor PlyGRCS activity. The influence of dose (FIG. 1, Plate A), pH (FIG.1, Plate B), NaCl (FIG. 1, Plate C), and divalent cations (FIG. 1, PlateD) on PlyGRCS activity against stationary phase S. aureus NRS-14 areshown. Error bars represent the standard deviation, and all experimentswere done in triplicate.

FIG. 2 are brightfield (FIG. 2, Plate A) and fluorescent (FIG. 2, PlateB) images showing SH3_5_(GRCS) directly interacting with S. aureusNRS-14. PlyGRCS contains a C-terminal cell wall binding domain. Cellwall binding was detected via Mouse Anti-His and Goat Anti-Mouse IgGAlexaFluor 488.

FIG. 3 illustrates PlyGRCS temperature stability. Stationary phase S.aureus NRS-14 treated with 25 μg/ml of PlyGRCS after being held atindicated temperatures for 30 min and recovered on ice for 5 min isshown in FIG. 3, Plate A. The thermal stability of full length PlyGRCS(FIG. 3, Plate B) as well as CHAP_(GRCS) (FIG. 3, Plate C) andSH3_5_(GRCS) (FIG. 3, Plate D) was determined by means of circulardichroism (CD) melting experiments. Samples were heated from 20° C. to95° C. at 1° C./min in 20 mM sodium phosphate buffer pH 7 using aprotein concentration of 0.1 mg/ml.

FIG. 4 illustrates antibiofilm activity of PlyGRCS. S. aureus NRS-14 wasallowed to form static biofilms for 24 hours and treated with PlyGRCS atindicated concentrations for 1 hour. The amount of biofilm isrepresented by the quantification of crystal violet staining of biomassat OD₅₉₅. Error bars represent the standard deviation, and allexperiments were done in triplicate.

FIG. 5 illustrates stationary phase S. aureus NRS-14 treated with 25μg/ml PlyGRCS, PlyGRCS-C29S, or PlyGRCS-H92A. PlyGRCS contains anN-terminal catalytic domain with an active-site cysteine and histidine.The reduction in activity of PlyGRCS-C29S and PlyGRCS-H92A indicatesthat these are the active-site residues. Error bars represent thestandard deviation, and all experiments were done in triplicate.

FIG. 6 illustrates the catalytic mechanism of PlyGRCS. As shown in FIG.6, Plate A, ESI-MS analysis of PlyGRCS digested peptidoglycan results ina spectrum (top) containing a peak at m/z=702.35, indicating thatPlyGRCS possesses endopeptidase and amidase activities. This peak isabsent in peptidoglycan digested with a known N-acetylmuramoyl-L-alanineamidase (second spectrum), or undigested peptodiglycan (third spectrum).Double digest with PlyGRCS and CHAP-K (bottom spectrum) yields aspectrum identical to that of PlyGRCS alone. FIG. 6, Plate B, showsschematically the A₂QKG₅ fragment corresponding to the 702.35 peakgenerated by both an N-acetylmuramoyl-L-alanine amidase activity (blackarrows) and a D-alanyl-glycyl endopeptidase activity (white arrows). Asshown in FIG. 6, Plate C, PlyGRCS peptidoglycan digest data shows boththe A₂QKG₅ (702.35 m/z peak) and the larger, doubly charged A₄Q₂K₂G₁₀moiety (684.84 m/z peak).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Embodiments of the present invention relate to compositions, methods anddevices for preventing or treating disease or infection associated withor caused by gram-positive bacteria, such compositions, methods anddevices incorporating and/or utlizing isolated endolysin polypeptide(s)from the GRCS bacteriophase (Sunagar R et al. (2010) Bacteriophagetherapy for Staphylococcus aureus bacteremia in streptozotocin-induceddiabetic mice. Res Microbiol 161(10):854-60). As used herein, an“isolated” endolysin polypeptide(s) or nucleic acid encoding suchpolypeptide(s) are free or substantially free of material with whichthey are naturally associated such as other polypeptides or nucleicacids. Polypeptides and nucleic acid may be formulated or mixed withpharmaceutically acceptable carriers, diluents or adjuvants (e.g., suchas in pharmaceutical compositions and/or when used in methods oftreatment or therapy) and still be isolated.

The PlyGRCS endolysin(s) of the present invention displaysdose-dependent antimicrobial activity against both planktonic andbiofilm S. aureus, including MRSA. The host range for this enzymeincludes all S. aureus and S. epidermidis strains tested. In someimplementations, compositions and methods including PlyGRCS endolysin(s)exhibit activity against S. aureus and S. epidermidis strains, but notagainst other Gram-positive pathogens.

PlyGRCS contains an N-terminal cysteine, histidine-dependentamidohydrolase/peptidase (CHAP) catalytic domain and a C-terminalbacterial src-homology 3 (SH3_5) binding domain. The endolysinpolypeptide(s) of the present invention may be isolated from the GRCSbacteriophage, or prepared by recombinant or synthetic methods as knownin the art. Nucleic acid and amino acid sequences of embodiments of thepresent invention are presented below:

PlyGRCS Native DNA Sequence (unoptimized) (SEQ ID NO: 1): ATGAAATCACAACAACaAGCaAAAGAATGGATATATAAACATGAGGGTACTgGTGTTgACTTTGATGGTGCATATgGtTTTCAATGTATGGAcTTAGCtgTTGctTaTgTATAtTACATTACAGACGGTAAAGTTCGTATGTgGGGTAACGCCAAAGACGCTATTAATAATGACTTTAAAGGTTTAGCAACGGTGTATGAAAATACACCGAGCTTTAAACCTCAATTAGGTGACGTTGCTGTTTATACTAATTCTCAATATGGTCACATTCAATGTGTaATAAGTGGTAATTTAGATTATTATACATGtTTAGAGCaAAACTGGTTAGGTGGTGGGTTTGACGGTTGGGaaAAAGCAACAATAAGAACACATTATTATGACGGTGTAACACACTTTATTAGACCtAAATTTTcTGCTAGTAATAGTAATGTATTAGAAACATCAAAAGTAAATaCATTTGGAAATTGGaAACaAAACCAATACGGAACATATTACAGAAATGAAAATgcAACATTTACATGTGGAtTTTTACCAATATTTGCACGTGTaGGTAGTcCTAAAtTAAGTGAAcCTAATgGATAtTgGtTcCaAcCaAATGGTTATACAcCATAtgACGAAGTTTGTTTATCAGATGGACTAgTGTGGATTgGTTATAATTGGCAAgGaACACGTTAttatttaccagtgaGACAATGGAACGGTAAAACGGGTAATAGTTATAGCATTGGTTTACCCTGGGGGGTGTTCTCA TAAPlyGRCS Native DNA Sequence (unoptimized;including 6x His tag (underlined) added at theC-terminus) (SEQ ID NO: 2): ATGAAATCACAACAACaAGCaAAAGAATGGATATATAAACATGAGGGTACTgGTGTTgACTTTGATGGTGCATATgGtTTTCAATGTATGGAcTTAGCtgTTGctTaTgTATAtTACATTACAGACGGTAAAGTTCGTATGTgGGGTAACGCCAAAGACGCTATTAATAATGACTTTAAAGGTTTAGCAACGGTGTATGAAAATACACCGAGCTTTAAACCTCAATTAGGTGACGTTGCTGTTTATACTAATTCTCAATATGGTCACATTCAATGTGTaATAAGTGGTAATTTAGATTATTATACATGtTTAGAGCaAAACTGGTTAGGTGGTGGGTTTGACGGTTGGGaaAAAGCAACAATAAGAACACATTATTATGACGGTGTAACACACTTTATTAGACCtAAATTTTcTGCTAGTAATAGTAATGTATTAGAAACATCAAAAGTAAATaCATTTGGAAATTGGaAACaAAACCAATACGGAACATATTACAGAAATGAAAATgcAACATTTACATGTGGAtTTTTACCAATATTTGCACGTGTaGGTAGTcCTAAAtTAAGTGAAcCTAATgGATAtTgGtTcCaAcCaAATGGTTATACAcCATAtgACGAAGTTTGTTTATCAGATGGACTAgTGTGGATTgGTTATAATTGGCAAgGaACACGTTAttatttaccagtgaGACAATGGAACGGTAAAACGGGTAATAGTTATAGCATTGGTTTACCCTGGGGGGTGTTCTCA CATCATCATCATCATCATTAAPlyGRCS Codon Optimized DNA Sequence (75%similarity to native DNA sequence) (SEQ ID NO: 3): ATGAAATCACAGCAGCAGGCTAAAGAATGGATTTATAAACATGAAGGAACTGGTGTTGATTTCGACGGCGCTTACGGGTTTCAGTGTATGGACCTGGCCGTGGCGTATGTGTACTATATTACCGACGGGAAAGTCCGTATGTGGGGTAATGCGAAGGATGCGATTAATAACGATTTTAAAGGCTTAGCCACGGTCTATGAAAATACTCCGTCATTTAAGCCGCAGCTGGGGGACGTGGCCGTATATACGAACAGCCAGTATGGGCATATCCAGTGCGTGATTAGCGGAAATCTGGACTACTACACGTGCCTTGAACAGAACTGGCTCGGGGGAGGGTTCGACGGTTGGGAAAAAGCGACTATCCGTACCCATTATTACGATGGAGTGACCCATTTTATTCGTCCGAAGTTTAGTGCTTCTAACAGCAATGTTCTGGAAACTAGCAAGGTGAATACTTTTGGAAACTGGAAACAGAATCAGTACGGCACGTATTATCGGAATGAGAACGCCACTTTCACGTGTGGTTTCCTGCCGATTTTCGCTCGTGTCGGCTCGCCTAAATTGTCCGAACCGAACGGCTATTGGTTCCAGCCGAATGGTTATACCCCGTATGATGAGGTGTGCTTGTCCGACGGTCTGGTGTGGATCGGTTACAACTGGCAGGGAACCCGTTACTACCTTCCGGTGCGTCAGTGGAATGGCAAAACGGGGAATTCTTACTCTATTGGACTTCCATGGGGCGTTTTTTCA TAAPlyGRCS Codon Optimized DNA Sequence (75%similarity to native DNA sequence; 6X His tagadded at the C-terminus) (SEQ ID NO: 4): ATGAAATCACAGCAGCAGGCTAAAGAATGGATTTATAAACATGAAGGAACTGGTGTTGATTTCGACGGCGCTTACGGGTTTCAGTGTATGGACCTGGCCGTGGCGTATGTGTACTATATTACCGACGGGAAAGTCCGTATGTGGGGTAATGCGAAGGATGCGATTAATAACGATTTTAAAGGCTTAGCCACGGTCTATGAAAATACTCCGTCATTTAAGCCGCAGCTGGGGGACGTGGCCGTATATACGAACAGCCAGTATGGGCATATCCAGTGCGTGATTAGCGGAAATCTGGACTACTACACGTGCCTTGAACAGAACTGGCTCGGGGGAGGGTTCGACGGTTGGGAAAAAGCGACTATCCGTACCCATTATTACGATGGAGTGACCCATTTTATTCGTCCGAAGTTTAGTGCTTCTAACAGCAATGTTCTGGAAACTAGCAAGGTGAATACTTTTGGAAACTGGAAACAGAATCAGTACGGCACGTATTATCGGAATGAGAACGCCACTTTCACGTGTGGTTTCCTGCCGATTTTCGCTCGTGTCGGCTCGCCTAAATTGTCCGAACCGAACGGCTATTGGTTCCAGCCGAATGGTTATACCCCGTATGATGAGGTGTGCTTGTCCGACGGTCTGGTGTGGATCGGTTACAACTGGCAGGGAACCCGTTACTACCTTCCGGTGCGTCAGTGGAATGGCAAAACGGGGAATTCTTACTCTATTGGACTTCCATGGGGCGTTTTTTCA CACCACCACCACCATCATTAAPlyGRCS Protein Sequence (amino acids 1-250) (SEQ ID NO: 5): MKSQQQAKEWIYKHEGTGVDFDGAYGFQCMDLAVAYVYYITDGKVRMWGNAKDAINNDFKGLATVYENTPSFKPQLGDVAVYTNSQYGHIQCVISGNLDYYTCLEQNWLGGGFDGWEKATIRTHYYDGVTHFIRPKFSASNSNVLETSKVNTFGNWKQNQYGTYYRNENATFTCGFLPIFARVGSPKLSEPNGYWFQPNGYTPYDEVCLSDGLVWIGYNWQGTRYYLPVRQWNGKTGNSYSIGLPWGVFSPlyGRCS Catalytic Domain (CHAP_(GRCS), amino acids1-140) (SEQ ID NO: 6): MKSQQQAKEWIYKHEGTGVDFDGAYGFQCMDLAVAYVYYITDGKVRMWGNAKDAINNDFKGLATVYENTPSFKPQLGDVAVYTNSQYGHIQCVISGNLDYYTCLEQNWLGGGFDGWEKATIRTHYYDGVTHFIRPKFSASPlyGRCS Binding Domain (SH3_5_(GRCS), amino acids150-250) (SEQ ID NO: 7): NTFGNWKQNQYGTYYRNENATFTCGFLPIFARVGSPKLSEPNGYWFQPNGYTPYDEVCLSDGLVWIGYNWQGTRYYLPVRQWNGKTGNSYSIGLPWGVFS

A “polypeptide” includes a polymer molecule comprised of multiple aminoacid residues joined in a linear manner. The polypeptide may includeconservative substitutions where the naturally occurring amino acid(s)is replaced by one having similar properties, where such conservativesubstitutions do not alter the function of the polypeptide.

The disclosed endolysin polypeptides may be engineered through domainshuffling or used in combination with other endolysins or antibiotics toprolong therapeutic efficacy (Shen Y et al. (2012) Phage-basedEnzybiotics. In: Abedon S, Hyman P (eds) Bacteriophages in Health andDisease. CABI Press, pp 217-239). Endolysin polypeptides of the presentinvention may be truncated, chimeric, shuffled or natural (e.g.,corresponding to wild-type). A “chimeric” polypeptide may be produced bycombining two or more proteins having two or more active sites. Chimericpolypeptides may act independently on the same or different molecules,and hence may potentially exhibit activity against two or more differentbacterial species or antigen targets.

In accordance with some embodiments, polypeptides are prepared orengineered to exhibit amino acid sequence percent identity of at least60%, 70%, 80%, 85%, and preferably at least 90%, 95%, 98% or 99% percentidentity, with active regions of PlyGRCS endolysin, including in SEQ IDNOs: 5, 6 and/or 7, and also exhibiting functionality and/or comparabletherapeutic efficacy (e.g., bacterial effects) therewith. Amino acidsequence percent identity is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the wild-type bacteriophage associated PlyGRCS endolysinsequence, after aligning the sequences in the same reading frame andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity.

Mutations can be made in the disclosed amino acid sequences, or in thenucleic acid sequences encoding the polypeptides herein, or in activefragments or truncations thereof, such that a particular codon ismodified to a codon which codes for a different amino acid, an aminoacid is substituted for another amino acid, or one or more amino acidsare deleted. Preferably, any such mutations do not significantly alterthe activity of the resulting polypeptide.

Thus, one of skill in the art, based on a review of the disclosedsequences of the PlyGRCS endolysin(s) of the present invention, mayimplement amino acid mutations in the polypeptide sequences to identifyadditional variants thereof (e.g., via random mutagenesis or by asite-directed method such as polymerase chain-mediated amplificationwith primers that encode the mutated locus). Further, mutagenizingentire codons rather than single nucleotides results in asemi-randomized repertoire of amino acid mutations. Libraries can beconstructed consisting of a pool of variants each of which differs by asingle amino acid alteration and/or which contain variants representingeach possible amino acid substitution for each residue. Variants may bescreened for desired activity using any screening method known in theart.

Variants may include one or more amino acid mutations (e.g., 1, 1-5,1-10, or 10 or more) in the sequence of the endolysin polypeptide(s),and also exhibit comparable functionality (e.g., comparable activityagainst bacteria) to the native endolysin polypeptide. Activity of suchvariant(s) may be tested using assays and methods as described hereinand as known in the art. One of skill in the art may predict suitableamino acid mutations to achieve such variants based on the disclosureherein.

As discussed in further detail below, contributions of the PlyGRCSputative catalytic and cell wall binding domains were investigatedthrough deletion analysis. PlyGRCS contains a putative cysteine,histidine-dependent amidohydrolase/peptidase (CHAP) catalytic domain.The CHAP domain alone displayed reduced (about 10%) activity as comparedto the full length protein, thus indicating that while this domain isresponsible for catalytic activity, the binding domain may be desirablein some applications for enhanced efficacy. In contrast, the SH3_5binding domain lacked activity but was shown to interact directly withthe staphylococcal cell wall via fluorescent microscopy.

Site-directed mutagenesis studies determined that active-site residuesin the CHAP catalytic domain were C29 and H92, with catalyticfunctionality benefiting from calcium as a co-factor. A decrease inactivity was observed, indicating the importance of these two residuesand the presence of an active CHAP domain.

Contributions of the putative catalytic and binding domains wereinvestigated through deletion analysis by turbidity reduction assay. TheCHAP catalytic domain displayed activity, though reduced and thusindicating advantages of also providing the binding domain for fullefficacy as noted above. The binding domain was confirmed byvisualization of cell wall interaction via fluorescent microscopy.Further, as determined by biochemical assays and mass spectrometry,PlyGRCS possesses an N-acetylmuramoyl-L-alanine amidase and aD-alanyl-glycyl endopeptidase catalytic mechanism.

Biochemical assays coupled with mass spectrometry analysis determinedthat PlyGRCS displays both N-acetylmuramoyl-L-alanine amidase andD-alanyl-glycyl endopeptidase hydrolytic activities despite possessingonly a single catalytic domain. Mass spectrometry of S. aureuspeptidoglycan digested by PlyGRCS showed a predominant peak at m/z=702,representative of dual catalytic activity. The results herein indicatethat PlyGRCS is a revolutionary therapeutic option to combat bacterialinfections.

PlyGRCS was found to exhibit strong activity against Staphylococcusspecies (e.g., such as S. aureus and S. epidermidis), and includingmethicillin- and vancomycin-resistant strains (e.g.,methicillin-resistant S. aureus (MRSA), vancomycinintermediate-resistant S. aureus (VISA), and methicillin-resistant S.epidermidis). Thus, the endolysin polypeptides of the present inventionwere demonstrated to be highly effective in killing, reducing oreliminating bacterial growth and/or population, and thus are suitablefor treating or preventing bacterial infection or symptoms associatedwith such bacteria in a subject (e.g., a human patient).

Compositions and methods utilizing or including the endolysinpolypeptide(s) of the present invention are effective in killing ortreating gram-positive bacteria in subjects, either alone or incomposition with one or more additional therapeutic agents, such as anantimicrobial or an antibiotic (e.g., including but not limited to, apenicillin, a cephalosporin, a polymyxin, an ansamycin, a quinolone, asulfonamide, a lipopeptide, a glycycline, and an oxazolidinone. In someimplementations, compositions or methods of treatment provide for theuse of PlyGRCS endolysin(s) in combination with one or more antibioticselected from linezolid, daptomycin, tigecycline, vancomycin,fidaxomicin, and/or metronidazole. In some implementations, theendolysin polypeptide(s) of the present invention, or therapeuticallyactive variants thereof, are covalently attached to an agent thatprovides additional functionality or enhances efficacy thereof. Suchagent(s) includes, for example, a tag, label, targeting moiety orligand, a cell binding motif or therapeutic agent, an antibacterial, anantibody, and an antibiotic.

Using turbidity reduction of stationary phase S. aureus as a measure oflytic activity, optimal conditions for PlyGRCS were determined, findingthat it is active in the physiological range. Compared to otherstaphylococcal endolysins, PlyGRCS is relatively active as only 25 μg/mLproduced a 70% decrease in optical density in just 15 minutes. Inaddition, its host range was characterized via plate lysis assays;PlyGRCS maintained lytic activity against all strains of S. aureustested as well as other staphylococcal species.

Further, crystal violet staining of PlyGRCS treated biofilmsdemonstrated that this enzyme is suitable for use on medical devices. Insome embodiments, the disclosed endolysin polypeptides and/orcompositions including the endolysin polypeptide(s) of the presentinvention are coupled to a surface of a substrate. For example, in someimplementations, a medical device (e.g., a grasper, a clamp, aretractor, a dilator, a suction, a sealing device, a scope, a probe,etc.) includes an outer surface coupled to or coated with the endolysinpolypeptide(s) or composition comprising the endolysin polypeptide(s) ofthe present invention. In some implementations, the medical devicecoupled to or coated with the disclosed endolysin polypeptide(s) orcomposition(s) is an implantable medical device (e.g., a drainage tube,a feeding tube, a shunt, a prosthesis, a guidance tube, a catheter, avalve, a pacemaker, a graft, a tissue scaffold, a stent, etc.).

The present invention provide for methods of treating a bacterialinfection in a patient comprising administering to the patient atherapeutically effective amount of an isolated endolysin polypeptide ofthe present invention, and in particular a polypeptide(s) comprising theamino acid sequence of SEQ ID NOs: 5, 6 and/or 7, or variants thereofsuch as a polypeptide(s) having at least 80% identity thereto andexhibiting comparable functionality and efficacy against bacteriaassociated with or causing said infection. The term “treat” or“treating” a disease, including an infectious disease or infection,refers to killing or reducing the growth of the bacteria causing suchdisease or infection, and/or reducing, ameliorating or eliminatingsymptoms associated with such disease or infection.

A “therapeutically effective amount” refers to the amount ofpolypeptide(s) sufficient to elicit a desired biological response in asubject, and in particular an amount sufficient to kill, reduce orstabilize a bacterial population causing such disease or infectionand/or sufficient to reduce symptoms associated with such disease orinfection. Preferably, a therapeutically effective amount of thepolypeptide(s) of the present invention is effective in reducing growthof the bacterial population by at least about 50%, more preferably by atleast about 75%, most preferably by about 90% or more.

The present invention is also directed to expression vectors preparedfrom the disclosed DNA sequences for expression in host systems, andencoding one or more of the endolysin polypeptide chains of the presentinvention. Such expression vectors may be used for recombinantproduction of the endolysin polypeptides of the invention. An expressionvector in the context of the present invention may be any suitable DNAor RNA vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40 bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors.

In one embodiment, the vector is suitable for expression of an endolysinpolypeptide of the present invention in a bacterial cell. Examples ofsuch vectors include expression vectors such as BlueScript (Stratagene),pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509 (1989),pET vectors (Novagen, Madison, Wis.), and the like. An expression vectormay also or alternatively be a vector suitable for expression in a yeastsystem. Any vector suitable for expression in a yeast system may beemployed. Suitable vectors include, for example, vectors comprisingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH (F. Ausubel et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley InterScience New York (1987); Grantet al., Methods in Enzymol 153, 516-544 (1987); Mattanovich, D. et al.Methods Mol. Biol. 824, 329-358 (2012); Celik, E. et al. Biotechnol.Adv. 30(5), 1108-1118 (2012); and Holliger, P. Methods Mol. Biol. 178,349-357 (2002)).

In an expression vector of the present invention, nucleic acids encodingthe disclosed polypeptides may comprise or be associated with anysuitable promoter, enhancer, and other expression-facilitating elements.Examples of such elements include strong expression promoters (e.g.,human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, andHIV LTR promoters), effective poly (A) termination sequences, an originof replication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE.

Vectors containing polynucleotides of interest can be introduced intothe host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell. Any host cell capable of overexpressing heterologous DNAscan be used for the purpose of isolating the genes encoding thepolypeptide or protein of interest, including for example, eukaryoticand prokaryotic hosts (e.g., strains of E. coli, Pseudomonas, Bacillus,Streptomyces, yeasts, etc.). As understood by those skilled in the art,not all vectors expression control sequences and hosts will functionequally well to express the DNA sequences of the present invention.However, those skilled in the art will be able to readily select theproper vectors, expression control sequences, and hosts to achieve thedesired expression.

The present invention provides for nucleic acids capable of encoding thedisclosed endolysin polypeptide(s). “Primer” as used herein refers to anoligonucleotide that is capable of acting as a point of initiation ofsynthesis when placed under suitable conditions in which synthesis of aprimer extension product is induced. The primer may be eithersingle-stranded or double-stranded and sufficiently long to prime thesynthesis of the desired extension product in the presence of aninducing agent. Exemplary primers are provided in Table 2 below.

The present invention also relates to pharmaceutical compositionscontaining therapeutically effective amounts of PlyGRCS endolysin(s)and/or variants and active fragments thereof. The pharmaceuticalcompositions may be formulated with pharmaceutically acceptable carriersor diluents as well as any other known adjuvants and excipients inaccordance with conventional techniques such as those disclosed inRemington: The Science and Practice of Pharmacy, 21th Edition, Gennaro,Ed., Mack Publishing Co., Easton, Pa., 2005.

The pharmaceutically acceptable carriers or diluents, as well as anyother known adjuvants and excipients, should be suitable for the chosencompound of the present invention and the chosen mode of administration.Suitability for carriers and other components of pharmaceuticalcompositions is determined based on the lack of significant negativeimpact on the desired biological properties of the chosen compound orpharmaceutical composition of the present invention (e.g., less than asubstantial impact (10% or less relative inhibition, 5% or less relativeinhibition, etc.)) on antigen binding.

A pharmaceutical composition of the present invention may thus includediluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in thecomposition. The diluent is selected to not affect the biologicalactivity of the combination. Examples of such diluents are distilledwater, physiological phosphate-buffered saline, Ringer's solutions,dextrose solution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, ornon-toxic, nontherapeutic, non-immunogenic stabilizers and the like. Thecompositions may also include large, slowly metabolized macromolecules,such as proteins, polysaccharides like chitosan, polylactic acids,polyglycolic acids and copolymers (e.g., latex functionalized sepharose,agarose, cellulose, and the like), polymeric amino acids, amino acidcopolymers, and lipid aggregates (e.g., oil droplets or liposomes).

The actual dosage levels of the active ingredient(s) in thepharmaceutical compositions of the present invention may be varied so asto obtain an amount of the active ingredient which is effective toachieve the desired therapeutic response for a particular patient,composition, and mode of administration. The selected dosage level willdepend upon a variety of pharmacokinetic factors including the activityof the particular compositions of the present invention employed, theroute of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the particular compositions employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

The pharmaceutical compositions of the present invention may beadministered by any suitable route and mode, including: parenteral,topical, oral or intranasal means for prophylactic and/or therapeutictreatment. In one embodiment, a pharmaceutical composition of thepresent invention is administered orally. In another embodiment, apharmaceutical composition of the present invention is administeredparenterally. The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includeepidermal, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrastemal injection and infusion.Additional suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the art.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound of thepresent invention. Examples of suitable aqueous and nonaqueous carrierswhich may be employed in the pharmaceutical compositions of the presentinvention include water, saline, phosphate buffered saline, ethanol,dextrose, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils,such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil,carboxymethyl cellulose colloidal solutions, tragacanth gum andinjectable organic esters, such as ethyl oleate, and/or various buffers.Other carriers are well known in the pharmaceutical arts and mayalternatively or additionally be included.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Pharmaceuticalcompositions of the present invention may also comprise isotonicityagents, such as sugars, polyalcohols, such as mannitol, sorbitol,glycerol or sodium chloride in the compositions.

Pharmaceutical compositions of the present invention may also containone or more adjuvants appropriate for the chosen route of administrationsuch as preservatives, wetting agents, emulsifying agents, dispersingagents, preservatives or buffers, which may enhance the shelf life oreffectiveness of the pharmaceutical composition.

The pharmaceutical compositions of the present invention may include asecondary therapeutic agent in addition to therapeutically effectiveamounts of the endolysin polypeptides disclosed herein, such as forexample an additional antimicrobial, antibiotic, and/or lytic enzyme.

The compounds of the present invention may be prepared with carriersthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Such carriers may includegelatin, glyceryl monostearate, glyceryl distearate, biodegradable,biocompatible polymers such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid aloneor with a wax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution and efficacy in vivo.Pharmaceutically acceptable carriers for parenteral administrationinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound(s), use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, microemulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe a aqueous or nonaqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In some cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe compositions may be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin. Sterile solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients e.g. as enumerated above, as required,followed by sterilization microfiltration. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients e.g. from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, examples ofmethods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Sterile solutions may be prepared by incorporating the active compoundin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time, or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. Pharmaceutical compositions may be formulated in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each unit contains apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and dependent on (a) thecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) any limitations in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

A physician having ordinary skill in the art may readily determine andprescribe the therapeutically effective amount of the pharmaceuticalcomposition required for a particular patient. Such amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe patient. In addition, the therapeutically effective amount is one inwhich any toxic or detrimental effects of the pharmaceutical compositionare outweighed by the therapeutically beneficial effects. The physicianmay start doses of the endolysin polypeptide(s) in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acomposition of the present invention will be that amount of the compoundwhich is the lowest dose effective to produce the desired therapeuticeffect (e.g., killing gram-positive bacteria, and in particularStaphylococcus species, e.g., S. aureus and S. epidermidis, andincluding methicillin- and vancomycin-resistant strains (e.g., MRSA,VISA, MRSE), and/or for treating or preventing infection, and/or forameliorating or alleviating symptoms associated with such bacteria in asubject). Such an effective dose will generally depend upon the factorsdescribed above. While it is possible for a compound of the presentinvention to be administered alone, it is preferable to administer thecompound as a pharmaceutical composition as described above.

Pharmaceutical compositions in accordance with the present invention maybe administered via spray, inhaler, topical, etc. Pharmaceuticalcompositions and polypeptides in accordance with disclosed embodimentsmay be administered via lozenges, chewing gums, tablets, powders,sprays, liquids, ointments, etc. Formulations including endolysinpolypeptides of the present invention may include additives,stabilizers, buffers, etc. as described above.

While some embodiments are described with respect to use in humans, theendolysin polypeptides, compositions and methods of the presentinvention are also suitable for veterinary (non-human) applications. Forexample, S. aureus is one of the most common causes of bovine mastitisin milking cows and prevention and control of such infection isdifficult. Once established, S. aureus infections do not respond well toconventional antibiotic therapy, and infected cows or other livestockmust often be segregated or culled from the herd. The spread of suchinfection within a group of livestock may occur through, inter alia,human contact (e.g., milkers' hands), equipment for maintaining andprocessing the animals, and flies. Thus, the polypeptide(s) of thepresent invention may be utilized for treating bacterial infection orcontamination in livestock or other animals (e.g., by administering thepolypeptide(s) of the present invention to such livestock or animalorally, nasally, parenternally, onto the skin or coat, via intramammaryinfusion, teat dip, etc. as described herein).

The endolysin polypeptides of the present invention, and compositionscomprising such polypeptides, are also suitable for use as a sanitizingagent or disinfectant of a target surface or area. Thus, the presentinvention provides for methods and compositions for treating orpreventing bacterial contamination of dental and medical devices,surfaces in hospitals and dental and medical facilities, food processingequipment, surfaces in food processing facilities, equipment andsurfaces in schools, and other equipment or surfaces on whichsanitization is desired.

In addition, the compositions of the present invention may be used incombination with other disinfecting ingredients, cleaners, and agents(e.g., such as detergents, solvents, antibiotics, antimicrobials, etc.).In some implementations, endolysin polypeptide(s) and compositions ofthe present invention are applied to target surfaces or areas as aliquid or spray formulation (e.g., aerosolized or mist formulation).Disclosed compositions may be applied, e.g., with a dry mist fogger orother such application, for disinfecting surfaces within a target areaor volume (e.g., a milking parlor, school gymnasium or auditorium,surgical suite, medical equipment, etc.).

Additional characteristics and features of the present invention will befurther understood through reference to the following examples anddiscussion, which are provided by way of further illustration and arenot intended to be limiting of the present invention.

Materials and Methods

Bacterial Strains.

Tested bacterial species, strains, and associated antimicrobialresistance phenotypes are shown in Table 1 below.

TABLE 1 PlyGRCS Host Range Resis- Bacterial species, strains tested¹tance² PlyGRCS³ CHAP_(GRCS) Staphylococcus aureus, NRS-385 MRSA + −Staphylococcus aureus, NRS-382 MRSA + − Staphylococcus aureus, NRS-384MRSA + + Staphylococcus aureus, NRS-71 MRSA ++ + Staphylococcus aureus,NRS-14 VISA ++ − Staphylococcus epidermidis, MRSE ++ + NRS-101Streptococcus suis, 730082 − − Streptococcus pyogenes, D471 − −Streptococcus pneumoniae, ATCC − − BAA-334 Streptococcus uberis, ATCC700407 − − Streptococcus equi, ATCC 9528 − − Bacillus pumulis, BJ0055 −− Enterococcus facealis, EF24 − − ¹See below for source of strains.²MRSA: methicillin-resistant S. aureus; VISA: vancomycinintermediate-resistant S. aueus; MRSE: methicillin-resistant S.epidermidis. ³Activity of 6 μg PlyGRCS or CHAP_(GRCS) was evaluated viaplate lysis assays. The strength of lytic zones was definedqualitatively: strong lytic zone = ++, weak lytic zone = +, no lyticzone = −.

All staphylococci containing the NRS strain designations were providedby the Network on Antimicrobial Resistance in S. aureus (NARSA), whichis distributed by BEI Resources depository (Manassas, Va.) under thedirection of the National Institute of Allergy and Infectious Diseasesand the National Institutes of Health. A Streptococcus suis clinicalisolate was obtained from Dr. Randy Shirbroun at Newport Laboratories(Worthington, Minn.). Streptococcus pyogenes and Enterococcus facealiswere obtained from Drs. Vincent Fischetti and Alexander Tomasz,respectively, at The Rockefeller University, USA). A Bacillus pumulisclinical isolate was obtained from Dr. John Mayo at Louisiana StateUniversity, USA. The remaining strains, Streptococcus pneumonia,Streptococcus uberis, and Streptococcus equi, were obtained from theAmerican Type Culture Collection (ATCC).

All strains were stored at −80° C. and routinely grown at 37° C.Streptococcal strains were grown in Todd-Hewitt broth, supplemented with1% yeast extract (THY) (Alpha Bioscience), or on THY plates;staphylococcal strains, B. pumulis, and E. facealis, were grown intrypticase soy broth (TSB) (Becton-Dickinson), or on TSB plates;Escherichia coli was cultivated in Luria Broth (LB) (Alpha Bioscience),or on LB plates. Chemicals were purchased from Sigma and were of thehighest purity available.

Cloning, Domain Constructs, and Site-Directed Mutagenesis.

The phage GRCS genome has recently been elucidated (GenBank AccessionKJ210330) (Swift S M & Nelson D C (2014) Complete genome sequence ofStaphylococcus aureus phage GRCS. Genome Announc 2(2)). Bioinformaticanalysis using BLAST and PFAM programs [National Center forBiotechnology Information (NCBI)] predicted a putative endolysin forORF15 (AHJ10590), referred to as PlyGRCS. As noted above, PlyGRCScontains an N-terminal cysteine, histidine-dependentamidohydrolase/peptidase (CHAP) catalytic domain and a C-terminalbacterial src-homology 3 (SH3_5) binding domain.

Individual domain clones for CHAP (i.e. CHAP_(GRCS)) and SH3_5 (i.e.SH3_5_(GRCS)) were amplified using the primer pairs shown in Table 2.

TABLE 2 Primers  Primer  Sequence CHAP-F5′-GGGGAATTCATTATGAAATCACAACAACAAGCAAAAGAATGGATATA-3′ (SEQ ID NO: 8)CHAP-R 5′-AAATCTAGATTAATGATGATGATGATGATGACTAGCAGAAAATTTAG-3′(SEQ ID NO: 9) SH3_55′-GGGGAATTCATTATGAATACATTTGGAAATTGGAAACAAAACCAATAC-3′ (SEQ ID NO: 10)SH3_5R 5′-AAATCTAGATTAATGATGATGATGATGATGTGAGAACACCCCCCAAG-3′(SEQ ID NO: 11) C29S 5′-[Phos]-GCATATGGTTTTCAAAGCATGGACTTAGCTGTT-3′(SEQ ID NO: 12) H92A 5′-[Phos]-AATTCTCAATATGGTGCGATTCAATGTGTAATA-3′(SEQ ID NO: 13)

For the full-length PlyGRCS, the CHAP-F and SH3 SR primers wereutilized. All reverse primers incorporated a 6×His purification tag.Specific point mutations to putative active-site residues (C29S andH92A) were made with phosphorylated primers (Table 2) using theChange-IT Multiple Mutation Site Directed Mutagenesis Kit (AffymetrixUSB) according to the manufacturer's instructions. All PCR products werecloned into pBAD24, transformed into E. coli BL21 (DE3) cells andconsequently sequenced (Macrogen, Rockville, Md.). The ApE program(University of Utah) was utilized for DNA sequence analysis andmanipulations.

Expression and Purification.

E. coli were grown at 37° C. in baffled flasks to an OD₆₀₀=1 in LBsupplemented with 100 μg/ml ampicillin. Expression was induced with0.25% arabinose overnight at 18° C. Crude protein extracts were purifiedby a Bio-Scale Mini Profinity IMAC Cartridge (Bio-Rad) and eluted in 10ml fractions of 20 mM, 50 mM, 100 mM, 250 mM, and 500 mM imidazole,followed by SDS-PAGE analysis. Fractions containing proteins of thecorrect predicted molecular weight were pooled and dialyzed against PBSpH 7.4 with 300 mM NaCl.

Quantification of Lytic Activity.

Lytic activity was based on turbidity reduction assay (Nelson D C et al.(2012) Endolysins as antimicrobials. Adv Virus Res 83:299-365). Briefly,bacterial cells were centrifuged (4,000 RPM, 5 minutes, 4° C.),re-suspended in buffer and mixed 1:1 (v/v) with endolysin to a finalOD₆₀₀=1. OD₆₀₀ readings were taken every 15 seconds for 20 minutes at37° C. Endolysin activity was equated to the V_(max) dictated by thelinear portion of the resulting killing curve. Each experiment wasperformed in triplicate.

Characterization of PlyGRCS.

To determine dose response, PlyGRCS was serially diluted and each dosage(100 μl) was added in triplicate to a 96-well polystyrene microtiterplate (Nest Biotech Co, Ltd) just before addition of bacterial cells(100 μl) according to the turbidity reduction assay described above. Foroptimum pH determination, bacterial cells were suspended in 40 mM boricacid/phosphoric acid (BP) buffer, pH 3-11, and were challenged againstPlyGRCS. The influence of NaCl on PlyGRCS activity was tested in BPbuffer at the experimentally determined pH optimum using the same assay.The effect of divalent cations was determined using the turbidityreduction assay with several modifications. First, PlyGRCS was incubatedat room temperature in PBS or PBS supplemented with 5 mM EDTA for 10minutes. Second, the EDTA-treated samples received either no furthertreatment, or were supplemented with 6 mM CaCl₂ or 6 mM MgCl₂. Finally,the lytic active of the samples was then immediately assayed andcompared to PlyGRCS in PBS prior to EDTA inactivation. Kinetic stabilitywas evaluated (Son B et al. (2012) Characterization of LysB4, anendolysin from the Bacillus cereus-infecting bacteriophage B4. BMCMicrobiol 12:33) with minor modifications. Lytic assays were performedin optimal conditions after PlyGRCS was incubated at indicatedtemperatures for 30 minutes and subsequently recovered on ice for 5minutes.

Cell Wall Binding.

An overnight culture of S. aureus NRS-14 was concentrated 5× in BPbuffer and was incubated at room temperature with 10 μg SH3_5_(GRCS)containing the 6×His tag for 10 minutes. A control without SH3_5_(GRCS)was also utilized. The samples were washed with PBS and incubated for 10minutes at room temperature with 1 μl mouse anti-His antibody (GenScript). After washing with PBS, AlexaFluor-488 conjugated goatanti-mouse IgG (H+L) antibody (1 μl) (Invitrogen) was incubated withsamples for an additional 10 minutes. Samples were washed again with PBSbefore being visualized via fluorescence and bright field microscopy. AnEclipse 80i epifluorescent microscope workstation (Nikon) with X-Cite120 illuminator (EXFO) and Retiga 2000R CCD camera was used.NIS-Elements software (Nikon) was used for image analysis.

Spectrum of Lytic Activity.

The PlyGRCS spectrum of lytic activity was performed (Schmelcher M etal. (2012) Chimeric phage lysins act synergistically with lysostaphin tokill mastitis-causing Staphylococcus aureus in murine mammary glands.Appl Environ Microbiol 78(7):2297-305) with minor modifications.Bacterial cells were diluted in sterile PBS to an OD₆₀₀=1, and 100 μlwas spread on each plate. 10 μl spots (600 μg/ml) of PlyGRCS orCHAP_(GRCS) were applied. Plates were incubated overnight at 37° C.Strength of lytic zones was defined qualitatively.

Biofilm Assay.

An overnight culture of S. aureus NRS-14 (1 ml per well) was placed into24-well CELLBIND plates (Corning) containing 500 μl of TSB per well.After an additional 24 hour incubation at 37° C., media was aspiratedand samples were washed with PBS to remove unattached cells. Two-foldserial dilutions of PlyGRCS in triplicate were added in 1 ml BP bufferpH 7 and incubated at 37° C. for one hour. Liquid was aspirated andsamples were washed with distilled water before drying. Biofilms werestained with 0.01% crystal violet for 10 min at room temperature. Afterremoving the excess crystal violet, samples were washed with PBS anddried before the addition of 1 ml 10% SDS to extract the crystal violetfrom the biomass for quantification at OD₅₉₅.

Bactericidal Analysis.

Sterile-filtered PlyGRCS was 2-fold serially diluted in PBS supplementedwith 1 mM CaCl₂ and an equal volume of either various concentrations ofenzyme or buffer only was added to 10⁵ S. aureus NRS-14 in a microtiterplate. Samples were incubated at 37° C. for 1 hour, then seriallydiluted, plated on TSB agar, and incubated overnight at 37° C. to obtainCFU counts. The MBC (minimum bactericidal concentration) was determinedas the minimum concentration of enzyme that killed ≥99.9% of bacteria.

Circular Dichroism (CD) Spectropolarimetry.

CD experiments for wild-type (WT) and active-site mutants were performedon a Chirascan CD spectrometer (Applied Photophysics) equipped with athermoelectrically controlled cell holder. CD spectra were obtained inthe far-UV range (190-260 nm) in a 1 mm path length quartz cuvette at 1nm steps with 5 second signal averaging per data point. Spectra werecollected in triplicate, followed by averaging, baseline subtraction,smoothing and conversion to mean residue ellipticity (MRE) by thePro-Data software (Applied Photophysics). Secondary structure predictionwas performed using the Provencher and Glockner method (Provencher S W &Glockner J (1981) Estimation of globular protein secondary structurefrom circular dichroism. Biochemistry 20(1):33-7) provided by DICHROWEB(Whitmore L & Wallace B A (2004) DICHROWEB, an online server for proteinsecondary structure analyses from circular dichroism spectroscopic data.Nucleic Acids Res 32:W668-73).

Melting experiments were performed by heating PlyGRCS at a 0.1 mg/mlconcentration in 20 mM sodium phosphate buffer pH 7 from 20° C. to 95°C. using a 1° C./min heating rate. MRE was monitored at 218 nm in a 1 mmpath length quartz cuvette at 0.5° C. steps with 5 second signalaveraging per data point. The melting data was smoothed, normalized andfit with a Boltzmann sigmoidal curve. The first derivative of themelting curve was then taken to determine the temperature (T_(m)) atwhich the folded and unfolded protein species in solution were atequilibrium (Fallas J A & Hartgerink J D (2012) Computational design ofself-assembling register-specific collagen heterotrimers. Nat Commun3:1087).

Biochemical Assays.

For analysis of reducing sugars released from the peptidoglycan, thedinitrosalicylic acid (DNSA) assay was used (Danner M et al. (1993)Folding and assembly of phage P22 tailspike endorhamnosidase lacking theN-terminal, head-binding domain. Eur J Biochem 215(3):653-61). S. aureusNRS-14 peptidoglycan was purified (Pritchard D G et al. (2004) Thebifunctional peptidoglycan lysin of Streptococcus agalactiaebacteriophage B30. Microbiology 150(Pt 7):2079-87; Schmelcher M et al.(2012) Listeria bacteriophage peptidoglycan hydrolases feature highthermoresistance and reveal increased activity after divalent metalcation substitution. Appl Microbiol Biotechnol 93(2):633-43), andtreated for one hour at 37° C. with 50 μg/ml of PlyGRCS in optimalconditions. Samples were centrifuged and the supernatant was added to anequal volume of 87.7 mM DNSA (20 g/L in 0.7 M NaOH). After boiling for 5min, samples were allowed to cool and the absorbance was read at OD₅₃₅.Known concentrations of glucose were used to create a standard curve. Todetermine an increase in free amine groups, the trinitrophenylationreaction was used, originally described by Satake et al. and modified byMokrasch (Satake M et al. (1960) Incorporation of leucine intomicrosomalprotein by a cell-free system of guinea-pig brain. BiochimBiophys Acta 41:366-7; Mokrasch L (1967) Use of2,4,6-trinitrobenzenesulfonic acid for the coestimation of amines, aminoacids, and proteins in mixtures. Anal Biochem 18:64-71). Purifiedpeptidoglycan (OD₆₀₀=1) was treated with PlyGRCS (50 μg/ml) for one hourat 37° C. Samples were pelleted and the supernatant was filtered througha 0.22 μM filter. The sterile filtrate was added to sodium tetraborateand trinitrobenzenesulfonic acid and incubated for 30 minutes at roomtemperature. Samples were read at OD₄₂₀. Lysine was used as a standard.

Cleavage Analysis by Mass Spectrometry.

For determination of cut sites within the staphylococcal peptidoglycan,purified cell walls were digested with PlyGRCS and the resultingfragments were analyzed via mass spectrometry (Becker S C et al. (2009)LysK CHAP endopeptidase domain is required for lysis of livestaphylococcal cells. FEMS Microbiol Lett 294(1):52-60; Pritchard D G etal. (2004) The bifunctional peptidoglycan lysin of Streptococcusagalactiae bacteriophage B30. Microbiology 150(Pt 7):2079-87). Briefly,SA113 ΔtagO cell walls (Atilano M L et al. (2010) Teichoic acids aretemporal and spatial regulators of peptidoglycan cross-linking inStaphylococcus aureus. Proc Natl Acad Sci USA 107(44):18991-6;Weidenmaier C et al. (2004) Role of teichoic acids in Staphylococcusaureus nasal colonization, a major risk factor in nosocomial infections.Nat Med 10(3):243-5) were digested in 25 mM Tris, 200 mM NaCl, pH 7.4 at37° C. for 18 hours with 50 μg/ml of PlyGRCS, filtered through 5000-MWcutoff Vivaspin 500 units (Sartorius North America Inc., Bohemia, N.Y.),and desalted using C18 Zip Tips (Millipore, Zug, Switzerland). Controlsincluded peptidoglycan digested with the amidase domain of 2638A, aknown N-acetylmuramoyl-L-alanine amidase, or undigested peptidoglycan.To further define the PlyGRCS cut site, double digests with PlyGRCS anda truncation construct containing only the CHAP domain of LysK (CHAP-K),a D-alany-glycyl endopeptidase were performed. The samples were elutedfrom the Zip Tips with 50:50:0.01 (v/v/v) CH₃OH:H₂O:HCOH (pH ˜2), andNanoESI-MS analysis was performed on a Q-TOF Ultima API massspectrometer (Micromass, UK).

Results

Expression of PlyGRCS and Domain Constructs.

The phage GRCS genome was recently sequenced (KJ210330) (Swift S M &Nelson D C (2014) Complete genome sequence of Staphylococcus aureusphage GRCS. Genome Announc 2(2)). Bioinformatic analysis predicted anendolysin for ORF15 (AHJ10590), referred to as PlyGRCS. This enzymecontains a putative N-terminal CHAP domain, which is shown to encompassbacteriolytic activity in other characterized endolysins, and aC-terminal bacterial src-homology 3 (SH3_5) domain that functions as aCBD in many staphylococcal and streptococcal endolysins (Nelson D C etal. (2012) Endolysins as antimicrobials. Adv Virus Res 83:299-365).

The closest homologs to PlyGRCS are a hypothetical protein from S.aureus 2011-60-1490-31 (EZV76040.1, 98% identity), an amidase fromStaphylococcus phage 44AHJD (NP_817310.1, 96% identity), ORF009 ofStaphylococcus phage 66 (YP_239469.1, 97% identity), the SAL-2 amidasefrom Staphylococcus phage SAP-2 (YP_001491539.1, 96% identity), and anunnamed protein product of Staphylococcus phage S24-1 (YP_004957430.1,92% identity). To study the full-length enzyme and elucidate thecontributions of each domain, the full length PlyGRCS was cloned, aswell as its isolated CHAP domain (CHAP_(GRCS), amino acids 1-140) andSH3_5 domain (SH3_5_(GRCS), amino acids 150-250) into expressionvectors. All three constructs were expressed as soluble proteins andpurified to homogeneity by nickel affinity chromatography via theC-terminal 6×His tags on each protein.

Characterization of PlyGRCS.

PlyGRCS displayed a dose response curve from 28 to 1.75 μg/ml whentested in a turbidity reduction assay using stationary phase S. aureusNRS-14 cells (FIG. 1, Plate A). The highest dose corresponded to a 70%decrease in optical density in just 15 minutes (50% decrease in <10minutes). When tested at equimolar concentrations, CHAP_(GRCS) displayed˜8-10% of full-length PlyGRCS activity. In contrast, SH3_5_(GRCS)displayed little to no lytic activity; however, this domain alonepossessed the ability to specifically bind staphylococci as detected byantibody recognition of the 6×His purification tag on the staphylococcalsurface (FIG. 2). Control experiments without SH3_5_(GRCS) diddemonstrate binding of the antibody. Therefore, while the CHAP domain isindependently capable of lysing S. aureus, for some applications,enhanced antimicrobial efficacy of the endolysin is provided by thesimultaneous presence of both the CHAP and SH3_5GRCS domains.

Lytic activity of PlyGRCS was then tested in BP buffer with a pH rangefrom 3.0 to 11.0 to determine optimum conditions. Optimal pH wasdetermined to be 7.0, with an active range between 6.0 and 8.0 (FIG. 1,Plate B). PlyGRCS activity was markedly reduced at pH extremes. Based onthe above observations, subsequent turbidity reduction and antimicrobialassays were performed in BP buffer pH 7.0. Because the activity of manyendolysins, including various staphylococcal endolysins (Becker S C etal. (2008) The phage K lytic enzyme LysK and lysostaphin actsynergistically to kill MRSA. FEMS Microbiol Lett 287(2):185-91; GarciaP et al. (2010) Synergy between the phage endolysin LysH5 and nisin tokill Staphylococcus aureus in pasteurized milk. Int J Food Microbiol141(3):151-5), is enhanced by the addition of NaCl, we investigated theactivity of PlyGRCS in the presence of NaCl ranging from 0 to 500 mM.Surprisingly, NaCl had little effect (±10%) on PlyGRCS activity up to125 mM and only slightly inhibited activity at higher concentrations(˜35% decrease at 500 mM) (FIG. 1, Plate C).

Several other CHAP domain-containing staphylococcal endolysins (DonovanD M et al. (2006) Lysis of staphylococcal mastitis pathogens bybacteriophage phi11 endolysin. FEMS Microbiol Lett 265(1):133-9; FentonM et al. (2011) Characterization of the staphylococcal bacteriophagelysin CHAP(K). J Appl Microbiol 111(4):1025-35), as well asstreptococcal endolysins (Celia L K et al. (2008) Characterization of abacteriophage lysin (Ply700) from Streptococcus uberis. Vet Microbiol130(1-2):107-17; Pritchard D G et al. (2004) The bifunctionalpeptidoglycan lysin of Streptococcus agalactiae bacteriophage B30.Microbiology 150(Pt 7):2079-87) have been shown to require calcium foractivity. Furthermore, the structure of the staphylococcal LysGH15 CHAPdomain shows calcium in an μF-hand-like structure (Gu J et al. (2014)Structural and biochemical characterization reveals LysGH15 as anunprecedented “EF-Hand-like” calcium-binding phage lysin. PLoS pathogens10(5):e1004109). The CHAP domain of PlyGRCS shares identity in threeaspartic acid residues known to complex this cation in LysGH15 and othercalcium-binding proteins, although it only shows 42% in overall identitywith the LysGH15 CHAP domain.

With this in mind, the activity of PlyGRCS was analyzed in either thepresence or absence of calcium. PlyGRCS was first incubated with EDTA toremove all divalent cations from solution. EDTA-treated PlyGRCS wasdevoid of lytic activity (FIG. 1, Plate D). Next, EDTA-treated PlyGRCSwas incubated with excess CaCl₂. Calcium-treated PlyGRCS displayednearly twice the lytic activity when compared to PlyGRCS prior to EDTAtreatment. To determine whether divalent metal dependence of PlyGRCS isspecific to calcium, the activity of the EDTA-treated endolysin wasmeasured after the addition of an alternative divalent metal, magnesium.The activity of magnesium-treated PlyGRCS mimicked that of theEDTA-treated sample, indicating that the divalent metal dependence ofPlyGRCS is calcium-specific.

Finally, the kinetic and thermodynamic stability of PlyGRCS wasinvestigated. PlyGRCS displayed >90% residual lytic activity afterincubating at temperatures ranging from 4° C. to 37° C. for a total of30 minutes. At temperatures of ≥40° C., lytic activity was not observed(FIG. 3, Plate A). Melting experiments performed on a CDspectrophotometer show cooperative unfolding of PlyGRCS with a T_(m) of43.5° C. (FIG. 3, Plate B), which further confirms the lack of activityat ≥40° C. observed during the kinetic stability experiment. CHAP_(GRCS)(FIG. 3, Plate C) and SH3_5_(GRCS) (FIG. 3, Plate D) had similar T_(m)values of 44.8° C. and 44.5° C., respectively. The observed PlyGRCSstability profile is consistent with that of other phage lysins. Forexample, the S. aureus endolysin LysK is kinetically inactivated at42.0° C. and the Streptococcus pneumoniae endolysin Cpl-1 displays atT_(m) of 43.5° C. (Filatova L Y et al. (2010) LysK, the enzyme lysingStaphylococcus aureus cells: specific kinetic features and approachestowards stabilization. Biochimie 92(5):507-13; Sanz J M et al. (1993)Thermal stability and cooperative domains of CPL1 lysozyme and itsNH2-and COOH-terminal modules. Dependence on choline binding. J BiolChem 268(9):6125-30).

PlyGRCS Spectrum of Lytic Activity.

In order to determine the spectrum of lytic activity of PlyGRCS,activity was tested against 13 different bacterial strains includingmethicillin-resistant and vancomycin-intermediate resistant S. aureus,methicillin-resistant S. epidermidis, and several other representativeGram-positive pathogens (see Table 1). At 6 lytic activity was seenagainst all staphylococcal strains, with PlyGRCS exhibiting the greateststrength against S. aureus strains NRS-71 and NRS-14 and S. epidermidisNRS-101. CHAP_(GRCS) exhibited less activity, causing relatively weakclearing zones on plates of S. aureus strains NRS-384 and NRS-71 and S.epidermidis NRS-101. Little to no lytic activity was observed on otherstrains. Thus, PlyGRCS has an activity spectrum relatively confined tostaphylococcal species, as little or no activity was observed againsttested streptococci or representative bacilli and enterococci species(Table 1).

Biofilm Assay.

Considering the ability of S. aureus to form biofilms and thus present afurther barrier to traditional treatments, we investigated theanti-biofilm properties of PlyGRCS. When 1 day biofilms were treatedwith PlyGRCS for 1 hour, a dose response decrease in the amount ofbiofilm was visualized, with as little as 6.25 μg/ml affecting a ˜50%decrease in biofilm biomass (FIG. 4).

Bactericidal Effects of PlyGRCS.

It has been noted that the minimal inhibitory concentration (MIC) assaymay not be the most appropriate assay to measure endolysin efficacy dueto the speed at which the enzyme acts (Kusuma C M et al. (2005)Comparison of four methods for determining lysostaphin susceptibility ofvarious strains of Staphylococcus aureus. Antimicrobial agents andchemotherapy 49(8):3256-63). Therefore, we employed the minimumbactericidal concentration (MBC) assay, which is the lowestconcentration of enzyme that kills ≥99.9% (i.e. 3 logs) of the testinoculum (Jones R et al. (1985) Susceptibility tests: microdilutionandmacrodilution broth procedures. In: Balows A, Hausler J, Shadomy H(eds) Manual of Clinical Microbiology. American Society forMicrobiology, Washington, DC, pp 972-7).

When tested against a VISA strain in stationary phase, 25 μg/ml PlyGRCSresulted in 3 log killing, 12.5 μg/ml yielded a 2.5 log decrease, and6.25 μg/ml reduced bacterial counts by 2 logs. Of note, VISA strainspossess thicker cell walls than other S. aureus strains. This phenotypemay cause the bacteria to be more resilient to endolysin treatment, andhence require higher than normal MBC values (Howden B P et al. (2010)Reduced vancomycin susceptibility in Staphylococcus aureus, includingvancomycin-intermediate and heterogeneous vancomycin-intermediatestrains: resistance mechanisms, laboratory detection, and clinicalimplications. Clin Microbiol Rev 23(1):99-139; Sieradzki K et al. (2003)Alterations of cell wall structure and metabolism accompany reducedsusceptibility to vancomycin in an isogenic series of clinical isolatesof Staphylococcus aureus. J Bacteriol 185(24):7103-10). Nonetheless, theresults compare favorably to other anti-staphylococcal endolysins. Forexample, PlySs2 represents the only other staphylococcal endolysin withreported bactericidal activity against a VISA strain, requiring 128μg/ml to decrease the colony counts of mid-log phase cells by 2 logs(Gilmer et al. (2013) Novel bacteriophage lysin with broad lyticactivity protects against mixed infection by Streptococcus pyogenes andmethicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother57(6):2743-50).

Confirmation of N-Terminal CHAP Catalytic Domain.

By definition, CHAP domains contain two invariant residues, a cysteineand a histidine (Bateman A & Rawlings N. Dak. (2003) The CHAP domain: alarge family of amidases including GSP amidase and peptidoglycanhydrolases. Trends Biochem Sci 28(5):234-7; Rigden D J et al. (2003)Amidase domains from bacterial and phage autolysins define a family ofgamma-D,L-glutamate-specific amidohydrolases. Trends Biochem Sci28(5):230-4). The cysteine acts as a catalytic nucleophile and thehistidine may function as a general base to deprotonate the thiol groupof the cysteine.

To determine the contributions of these putative residues in PlyGRCS, weused site-directed mutagenesis to alter C29 and H92, the residuesidentified by a PFAM alignment of PlyGRCS to archetypical CHAP domains.Circular dichroism analysis demonstrated that both the C29S and H92Apoint mutants had similar secondary structures to WT PlyGRCS. No lyticactivity was observed when the C29S mutant was used against S. aureusNRS-14 in a turbidity reduction assay (FIG. 5); however, H92A stillexhibited lytic activity, although reduced to about 40% as compared toWT activity. Similar mutagenesis of active-site histidine residues incysteine proteases have likewise displayed reduced but measurableactivity (Ekici O D et al. (2008) Unconventional serine proteases:variations on the catalytic Ser/His/Asp triad configuration. Proteinscience: a publication of the Protein Society 17(12):2023-37; Khayat Ret al. (2001) Investigating the role of histidine 157 in the catalyticactivity of human cytomegalovirus protease. Biochemistry40(21):6344-51). Thus, other residues near the active-site residues maysubstitute for the histidine as an electron acceptor during thenucleophilic attack by the cysteine.

Cleavage Specificity of the CHAP Domain.

CHAP domains are associated with N-muramoyl-L-alanine amidase (amidase)or endopeptidase activity (Bateman A & Rawlings N. Dak. (2003) The CHAPdomain: a large family of amidases including GSP amidase andpeptidoglycan hydrolases. Trends Biochem Sci 28(5):234-7). Specifically,CHAP domains of staphylococcal endolysins have been characterized asamidases or D-alanyl-glycyl endopeptidases (Schmelcher M et al. (2012)Bacteriophage endolysins as novel antimicrobials. Future Microbiol7(10):1147-71). To determine the specific catalytic nature of thePlyGRCS CHAP domain, two biochemical assays were employed to analyze thereducing sugar (indicative of glycosidase activity) or amine (indicativeof amidase/endopeptidase activity) release upon PlyGRCS treatment. Asexpected, PlyGRCS did not show any glycosidase activity. However, freeamines were detected when S. aureus cell walls were treated withPlyGRCS, demonstrating that the catalytic activity is indeed an amidaseor endopeptidase.

To further elucidate which hydrolytic activity PlyGRCS possesses,enzymatically digested S. aureus peptidoglycan preparations weresubjected to electron spray ionization-mass spectrometry (ESI-MS).Unexpectedly, the PlyGRCS digest (FIG. 6, Plate A, top spectrum)revealed a peak at m/z=702.35, which could only be produced by thepresence of two enzymatic activities, an N-acetylmuramoyl-L-alanineamidase and either a D-alanyl-glycyl endopeptidase or a glycyl-glycylendopeptidase, to yield the fragment A₂QKG₅ (single letter amino acidcode) (FIG. 6, Plate B).

Moreover, a larger double-charged ion (m/z=684.84) was also observedthat likely corresponds to the fragment A₄Q₂K₂G₁₀ (without a watermolecule), resulting from incomplete peptidoglycan digest (FIG. 6,Plates A and C). Presence of the 702.35 and 684.84 peaks wasreproducible on independent digests and ESI-MS experiments. Controlexperiments with peptidoglycan digested with the 2638A amidase domain, aknown N-acetylmuramoyl-L-alanine amidase (FIG. 6, Plate A, secondspectrum), or undigested peptidoglycan (FIG. 6, Plate A, third spectrum)did not contain the 702.35 or 684.84 peaks indicating that generation ofthe 702.35 and 684.84 fragments by PlyGRCS was not an artifact of asingle enzymatic activity acting on uncrosslinked or partially cleavedpeptidoglycan.

Furthermore, a double digest with PlyGRCS and CHAP-K, which hasD-alanyl-glycyl endopeptidase activity (Becker S C et al. (2009) LysKCHAP endopeptidase domain is required for lysis of live staphylococcalcells. FEMS Microbiol Lett 294(1):52-60) was performed to elucidate thespecific nature of the endopeptidase activity. Because this spectrum wasidentical to that of the PlyGRCS alone digested peptidoglycan, it wasdetermined that PlyGRCS possesses a D-alanyl-glycyl endopeptidaseactivity, as a glycyl-glycyl endopeptidase activity would have yielded adifferent fragment pattern.

Thus, these data indicate that PlyGRCS, which has a single catalyticCHAP domain, can cleave two distinct bonds in the staphylococcalpeptidoglycan.

DISCUSSION

The use of endolysins provides a targeted treatment for bacterialinfections that circumvents traditional antibiotic resistance mechanisms(Sprott B G (1994) Resistance to antibiotics mediated by targetalterations. Science 264(5157):388-93). In accordance with embodimentsof the present invention, PlyGRCS endolysin was characterized anddemonstrated bacteriolytic activity against MRSA successfully. Theendolysin dosage used demonstrated that the efficacy of PlyGRCS iscomparable to or better than other published staphylococcal endolysins(Gilmer D B et al. (2013) Novel bacteriophage lysin with broad lyticactivity protects against mixed infection by Streptococcus pyogenes andmethicillin-resistant Staphylococcus aureus. Antimicrobial agents andchemotherapy 57(6):2743-50; Jun S Y et al. (2011) Comparison of theantibacterial properties of phage endolysins SAL-1 and LysK.Antimicrobial agents and chemotherapy 55(4):1764-7; Sass P & Bierbaum G(2007) Lytic activity of recombinant bacteriophage phi11 and phi12endolysins on whole cells and biofilms of Staphylococcus aureus. ApplEnviron Microbiol 73(1):347-52). Moreover, because the optimalconditions for PlyGRCS activity were determined to be in thephysiological range, this enzyme is suitable for use as an antimicrobialagent.

Even more impressive is the ability of PlyGRCS to act against stationaryphase staphylococci as well as medically relevant biofilms, a furtherhindrance to traditional antibiotic therapy. Given the ability ofendolysins like PlyGRCS to disrupt biofilms, they could be utilized inconjunction with classical antibiotics. In some embodiments, theendolysin provides the initial disturbance to the biofilm structure,thereby allowing the antibiotic to subsequently access the nowsusceptible target bacteria. Antibiotics applied in combination withendolysins bind more efficiently to their planktonic target bacterialcells, and thus this same phenomenon may be observed in biofilms as well(Schuch R et al. (2013) Combination therapy with lysin CF-301 andantibiotic is superior to antibiotic alone for treatingmethicillin-resistant Staphylococcus aureus-induced murine bacteremia. JInfect Dis 2014:209).

Identification of the PlyGRCS cleavage sites is also an importantfinding. This is believed to be the first demonstration of a single CHAPdomain, or any individual endolysin catalytic domain, that possesses theability to cleave two disparate bonds in the bacterial peptidoglycan.Initially, it was thought that the results were attributed to a singlecleavage of uncrosslinked peptidoglycan, resulting in a fragment thatappeared to be created by two cleavage events. However, spectra fromrepeated experiments on undigested control peptidoglycan and controldigests with enzymes of known specificity collectively indicate thatPlyGRCS is capable of liberating the fragment A₂QKG₅ from thestaphylococcal peptidoglycan. This would necessitate cleavage of theamide bond formed between MurNAc and Ala residues as well as thehydrolysis of the amide bond formed between D-Ala and Gly residues orone of the Gly-Gly bonds. Further experiments with a double digestincluding PlyGRCS and CHAP-K, a D-alanyl-glycyl endopeptidase, showed anidentical pattern to the PlyGRCS only spectrum, indicating that theendopeptidase activity of PlyGRCS is identical to CHAP-K.

While these findings indicating both amidase and endopeptidaseactivities associated with the single CHAP domain-containing PlyGRCSwere surprising, other CHAP domains have been associated with anN-acetylmuramoyl-L-alanine amidase activity in the streptococcal PlyCendolysin (McGowan S et al. (2012) X-ray crystal structure of thestreptococcal specific phage lysin PlyC. Proc Natl Acad Sci USA109(31):12752-7) as well as D-alanyl-glycyl endopeptidase activity inmultiple staphylococcal endolysins (Schmelcher M et al. (2012)Bacteriophage endolysins as novel antimicrobials. Future Microbiol7(10):1147-71). Moreover, the recently crystallized CHAP domain from thestaphylococcal endolysin LysGH15 shows highest structural homology tothe aforementioned CHAP domain of PlyC, with a root-mean-squaredeviation (RMSD)=2.32 Å (Gu J et al. (2014) Structural and biochemicalcharacterization reveals LysGH15 as an unprecedented “EF-Hand-like”calcium-binding phage lysin. PLoS pathogens 10(5):e1004109), furthersupporting the conclusion that these domains exhibit multipleactivities. Finally, consistent with the findings of our biochemicalassays, both amidase and endopeptidase activities would yield free aminegroups via cleavage of peptide moieties and additionally would notliberate reducing sugars, which requires the cleavage of at least one ofthe two glycosidic bonds responsible for maintaining the glycan backboneof peptidoglycan.

The implications of a single catalytic domain with two cleavagespecificities are numerous for bioengineering efforts. First, endolysinsdisplay synergy with other endolysins of different cleavagespecificities. For example, killing of pneumococci is enhanced when theendolysins Cpl-1, an N-acetylmuramidase, and PAL, anN-acetylmuramoyl-L-alanine amidase, are used together compared to twicethe concentration of either enzyme alone (Loeffler J M & Fischetti V A(2003) Synergistic lethal effect of a combination of phage lytic enzymeswith different activities on penicillin-sensitive and—resistantStreptococcus pneumoniae strains. Antimicrobial agents and chemotherapy47(1):375-7). Likewise, mutagenesis of active-site residues was used toshow synergy between two catalytic domains, anN-acetylmuramoyl-L-alanine amidase and a glycosyl hydrolase, within thePlyC endolysin (McGowan S et al. (2012) X-ray crystal structure of thestreptococcal specific phage lysin PlyC. Proc Natl Acad Sci USA109(31):12752-7). It is believed that synergy arises from cleaving thepeptidoglycan at two different locations, which is more destabilizing tothe superstructure than repetitive cleavages at one location and wouldresult in accelerated osmolysis of the bacterial cell. Additionally,cleavage of one bond may facilitate access to the second target, furthercontributing to this synergistic effect.

A second benefit of a catalytic domain with dual activities is that itis less susceptible to development of resistance. While there arecurrently no specific reports of bacterial strains developing resistanceto phage-encoded endolysins, resistance to peptidoglycan hydrolases as ageneral class has been reported. Notably, modifications to thepeptidoglycan backbone can render N-acetylmuramidases (i.e. lysozymes)ineffective (Davis K M & Weiser J N (2011) Modifications to thepeptidoglycan backbone help bacteria to establish infection. InfectImmun 79(2):562-70; Vollmer W (2008) Structural variation in the glycanstrands of bacterial peptidoglycan. FEMS Microbiol Rev 32(2):287-306).More specific to the staphylococcal peptidoglycan, resistance tolysostaphin, a bacterial derived glycyl-glycine endopeptidase, can beachieved by simple modification of the pentaglycine crossbridge in thesespecies (Nelson D C et al. (2012) Endolysins as antimicrobials. AdvVirus Res 83:299-365). Thus, endolysins engineered to have more than onecatalytic activity would circumvent resistance development targeting thespecificity of one activity. As protein therapeutics, PlyGRCS isamenable to domain shuffling, directed evolution, and bioengineeringapproaches to further enhance efficacy and/or specificity. The uniquedual substrate activity of the PlyGRCS catalytic domain thus offers anideal model for identifying other domains from staphylococcal-specificendolysins.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein its entirety. While the invention has been described in connectionwith exemplary embodiments thereof, it will be understood that it iscapable of further modifications and this application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure as come within known or customarypractice within the art to which the invention pertains and as may beapplied to the features hereinbefore set forth.

What is claimed is:
 1. A pharmaceutical composition for killingGram-positive bacteria comprising an isolated polypeptide comprising theamino acid sequence of SEQ ID NO: 6 or variants thereof having at least80% identity thereto and effective for killing said bacteria, and apharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1, wherein said isolated polypeptide further comprises theamino acid sequence of SEQ ID NO:
 7. 3. The pharmaceutical compositionof claim 1, wherein said isolated polypeptide comprises the amino acidsequence of SEQ ID NO:
 5. 4. The pharmaceutical composition of claim 1,further comprising one or more antibiotic.
 5. The pharmaceuticalcomposition of claim 4, wherein said antibiotic is selected from thegroup consisting of a penicillin, a cephalosporin, a polymyxin, anansamycin, a quinolone, a sulfonamide, a lipopeptide, a glycycline, andan oxazolidinone.
 6. The pharmaceutical composition of claim 4, whereinsaid antibiotic is selected from the group consisting of linezolid,daptomycin, and tigecycline, vancomycin, fidaxomicin, and metronidazole.7. An isolated polypeptide capable of killing one or more strain ofStaphylococcus bacteria, comprising the amino acid sequence of SEQ IDNO: 6 or variants thereof having at least 80% identity thereto andeffective for killing said bacteria.
 8. The isolated polypeptide ofclaim 7, further comprising the amino acid sequence of SEQ ID NO:
 7. 9.The isolated polypeptide of claim 7, which comprises the amino acidsequence of SEQ ID NO:
 5. 10. A nucleic acid encoding the isolatedpolypeptide of claim
 7. 11. A surface of a substrate comprising anantibacterial coating, said coating comprising an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 6 or variants thereofhaving at least 80% identity thereto.
 12. The surface of claim 11,wherein said isolated polypeptide further comprises the amino acidsequence of SEQ ID NO:
 7. 13. The surface of claim 11, wherein saidisolated polypeptide comprises the amino acid sequence of SEQ ID NO: 5.14. The surface of claim 11, wherein said coating further comprises oneor more antibiotic.
 15. The surface of claim 14, wherein said antibioticis selected from the group consisting of a penicillin, a cephalosporin,a polymyxin, an ansamycin, a quinolone, a sulfonamide, a lipopeptide, aglycycline, and an oxazolidinone.
 16. The surface of claim 11, whereinsaid substrate is a medical device.
 17. The surface of claim 11, whereinsaid substrate is an implantable medical device.